The present invention is directed to a process using microorganisms or enzymes derived therefrom for deacetylation of taxanes at C-4 to give 4-deacetyltaxanes, useful intermediates for the synthesis of new anti-cancer agents.
Taxanes are diterpene compounds which find utility in the pharmaceutical field. For example, taxanes containing aryl heterocyclic or cycloalkyl groups on the C-13 sidechain find utility as anti-cancer agents. Taxanes include pacltitaxel, cephalomannine, taxol c, 10-deacetylpaclitaxel, 10-deacetylcephalomannine, 7-xcex2-xylosylpaclitaxel, baccatin-III, 10-deacetylbaccatin III, 7-xcex2-xylosyl-10-deacetyl cephalomannine, 7-xcex2-xylosyl-10-deacetylbaccatin III, 7-xcex2-xylosylbaccatin III, and 10-deacetyl-taxol c.
Paclitaxel (Taxol), a diterpene taxane compound, is a natural product extracted from the bark of the Pacific yew tree, Taxus Brevifolia. It has been shown to have excellent antitumor activity in in vivo animal models, and recent studies have elucidated its unique mode of action, which involves abnormal polymerization of tubulin and disruption of mitosis during the cell cycle. Taxol has recently been approved for the treatment of refractory advanced ovarian cancer, breast cancer, non-small cell lung cancer, and most recently, AIDS-related Kaposi""s Sarcoma. The results of paclitaxel clinical studies are replete in scientific periodicals and have been reviewed by numerous authors, such as Rowinsky and Donehower in xe2x80x9cThe Clinical Pharmacology and Use of Antimicrotubule Agents in Cancer Chemotherapeuticsxe2x80x9d, Phamac. Ther., 52, pp. 35-84 (1991); Spencer and Faulds, Paclitaxel, A Review of its Pharmacodynamic and Pharmacokinetic Properties and Therapeutic Potential in the Treatment of Cancer, Drugs, 48 (5), pp. 794-847 (1994); K. C. Nicolau et al., Chemistry and Biology of Taxol, Angew. Chem., Int. Ed. Eng., 33, pp. 15-44 (1994); F. A. Holmes, A. P. Kudelka, J. J. Kavanaugh, M. H. Huber, J. A. Ajani, and V. Valero, xe2x80x9cTaxane Anticancer Agentsxe2x80x94Basic Science and Current Statusxe2x80x9d, edited by Gunda I. Georg, Thomas C. Chen, lwao Ojima, and Dolotrai M. Vyas, pp. 31-57 American Chemical Society, Wash., D.C. (1995); Susan G. Arbuck and Barbara Blaylock, xe2x80x9cTaxol( Science and Applicationsxe2x80x9d, edited by Matthew Suffness, pp. 379-416, CRC Press, Boca Raton, Fla. (1995) and the references cited therein.
The structure of Taxol(copyright) is shown below along with the conventional numbering system for molecules belonging to the Taxane class; such numbering system is also employed in this application. 
With reference to the numbering of the taxane, reference to a particular carbon on the taxane structure shall be indicated throughout this application by a xe2x80x9cC-numberxe2x80x9d, which signifies the carbon on the taxane according to the above numbering system. For example, xe2x80x9cC-13xe2x80x9d refers to the carbon at position 13 on the taxane ring as shown above, having a sidechain coupled thereto.
The central backbone structural unit of paclitaxel is baccatin III, a diterpenoid having the chemical structure: 
It is also very similar in structure to 10-deacetylbaccatin III (xe2x80x9c10-DAB IIIxe2x80x9d), which has the chemical structure: 
but which lacks an acetate ester at the 10-position alcohol.
Chemical modification of the paclitaxel structure at C-4 and other positions has been explored by many groups to determine structure/activity relationships and to try to obtain compounds with superior efficacy to taxol to develop as second generation drugs. See U.S. Pat. No. 5,773,461; Gunda I. George, Syed M. Ali, Thomas C. Boge, Apurba Datta, and Lise Falborg, xe2x80x9cSelective C-2 and C-4 Deacylation of Taxol: The First Synthesis of a C-4 Substituted Taxol Analoguexe2x80x9d, Tetrahedron Let., 35:48, pp. 8931-8934 (1994); Shu-Hui Chen, John F. Kadow, Vittorio Farina, Craig R. Fairchild and Kathy A. Johnston, xe2x80x9cFirst Synthesis of Novel Paclitaxel (Taxol) Analogs Modified at the C-4 Positionxe2x80x9d, J. Org. Chem. 59, pp. 6156-6158 (1994); S. Py, and F. Khuong-Huu, xe2x80x9cA Novel Rearrangement of The Taxane Skeletonxe2x80x9d, Bull. Soc. Chim. Fr., 130, pp. 189-191 (1993).
Replacement of the C-4-acetyl group of paclitaxel with other substituents has lead to compounds with improved potency in activity assays (S. Chen et al., Biorganic and Medicinal Chemistry Letters, 5:2741-2748 (1995)). An enzyme capable of specifically removing the C-4-acetyl group from taxanes will be useful in the synthesis of C-4-modified paclitaxel analogs to provide starting material to allow incorporation of other groups at this position, for example C-4 butyrate esters, C-4 cyclobutyl esters, C-4 propyl esters, C-4 cyclopropyl esters and C-4 methyl and ethyl carbonates.